Nav

Wednesday, January 25, 2012

It takes a lot of genes to wire the human brain.Billions of cells, of a myriad different types have to be specified, directed to migrate to the right position, organised in clusters or layers, and finally connected to their appropriate targets.When the genes that specify these neurodevelopmental processes are mutated, the result can be severe impairment in function, which can manifest as neurological or psychiatric disease.

How those kinds of neurodevelopmental defects actually lead to the emergence of particular pathological states – like psychosis or seizures or social withdrawal – is a mystery, however.Many researchers are trying to tackle this problem using mouse models – animals carrying mutations known to cause autism or schizophrenia in humans, for example.A recent study from my own lab (open access in PLoS One) adds to this effort by examining the consequences of mutation of an important neurodevelopmental gene and providing evidence that the mice end up in a state resembling psychosis.In this case, we start with a discovery in mice as an entry point to the underlying neurodevelopmental processes.

In just the past few years, over a hundred different mutations have been discovered that are believed to cause disorders like autism or schizophrenia.In many cases, particular mutations can actually predispose to many different disorders, having been linked in different patients to ADHD, epilepsy, mental retardation or intellectual disability, Tourette’s syndrome, depression, bipolar disorder and others.These clinical categories may thus represent more or less distinct endpoints that can arise from common neurodevelopmental origins.

For a condition like schizophrenia, the genetic overlap with other conditions does not invalidate the clinical category.There is still something distinctive about the symptoms of this disorder that needs to be explained.I have argued that schizophrenia can clearly be caused by single mutations in any of a very large number of different genes, many with roles in neurodevelopment.If that model is correct, then the big question is: how do these presumably diverse neurodevelopmental insults ultimately converge on that specific phenotype?It is, after all, a highly unusual condition.The positive symptoms of psychosis – hallucinations and delusions, for example – especially require an explanation.If we view the brain from an engineering perspective, then we can say that the system is not just not working well – it is failing in a particular and peculiar manner.

To try to address how this kind of state can arise we have been investigating a particular mouse – one with a mutation in a gene called Semaphorin-6A.This gene encodes a protein that spans the membranes of nerve cells, acting in some contexts as a signal to other cells and in other contexts as a receptor of information. It has been implicated in controlling cell migration, the guidance of growing axons, the specification of synaptic connectivity and other processes.It is deployed in many parts of the developing brain and required for proper development in the cerebral cortex, hippocampus, thalamus, cerebellum, retina, spinal cord, and probably other areas we don’t yet know about.

Despite widespread cellular disorganisation and miswiring in their brains, Sema6A mutant mice seem overtly pretty normal.They are quite healthy and fertile and a casual inspection would not pick them out as different from their littermates.However, more detailed investigation revealed electrophysiological and behavioural differences that piqued our interest.

Because these animals have a subtly malformed hippocampus, which looks superficially like the kind of neuropathology observed in many cases of temporal lobe epilepsy, we wanted to test if they had seizures.To do this we attached electrodes to their scalp and recorded their electroencephalogram (or EEG).This technique measures patterned electrical activity in the underlying parts of the brain and showed quite clearly that these animals do not have seizures.But it did show something else – a generally elevated amount of activity in these animals all the time.

What was particularly interesting about this is that the pattern of change (a specific increase in alpha frequency oscillations) was very similar to that reported in animals that are sensitised to amphetamine – a well-used model of psychosis in rodents.High doses of amphetamine can acutely induce psychosis in humans and a suite of behavioural responses in rodents.In addition, a regimen of repeated low doses of amphetamine over an extended time period can induce sensitisation to the effects of this drug in rodents, characterised by behavioural differences, like hyperlocomotion, as well as the EEG differences mentioned above.Amphetamine is believed to cause these effects by inducing increases in dopaminergic signaling, either chronically, or to acute stimuli.

This was of particular interest to us, as that kind of hyperdopaminergic state is thought to be a final common pathway underlying psychosis in humans. Alterations in dopamine signaling are observed in schizophrenia patients (using PET imaging) and also in all relevant animal models so far studied.

To explore possible further parallels to these effects in Sema6A mutants we examined their behaviour and found a very similar profile to many known animal models of psychosis, namely hyperlocomotion and a hyper-exploratory phenotype (in addition to various other phenotypes, like a defect in working memory).The positive symptoms of psychosis can be ameliorated in humans with a number of different antipsychotic drugs, which have in common a blocking action on dopamine receptors.Administering such drugs to the Sema6A mutants normalised both their activity levels and the EEG (at a dose that had no effect on wild-type animals).

These data are at least consistent with (though they by no means prove) the hypothesis that Sema6A mutants end up in a hyperdopaminergic state.But how do they end up in that state?There does not seem to be a direct effect on the development of the dopaminergic system – Sema6A is at least not required to direct these axons to their normal targets.

Our working hypothesis is that the changes to the dopaminergic system emerge over time, as a secondary response to the primary neurodevelopmental defects seen in these animals.

It is well documented that early alterations, for example to the hippocampus, can have cascading effects over subsequent activity-dependent development and maturation of brain circuits.In particular, it can alter the excitatory drive to the part of the midbrain where dopamine neurons are located, in turn altering dopaminergic tone in the forebrain.This can induce compensatory changes that ultimately, in this context, may prove maladaptive, pushing the system into a pathological state, which may be self-reinforcing.

For now, this is just a hypothesis and one that we (and many other researchers working on other models) are working to test.The important thing is that it provides a possible explanation for why so many different mutations can result in this strange phenotype, which manifests in humans as psychosis.If this emerges as a secondary response to a range of primary insults then that reactive process provides a common pathway of convergence on a final phenotype.Importantly, it also provides a possible point of early intervention – it may not be possible to “correct” early differences in brain wiring but it may be possible to prevent them causing transition to a state of florid psychopathology.

Sunday, January 8, 2012

Unlike in many other animals, injured nerve fibres in the mammalian central nervous system do not regenerate – at least not spontaneously.A lot of research has gone in to finding ways to coax them to do so, unfortunately with only modest success.The main problem is that there are many reasons why central nerve fibres don’t regenerate after an injury – tackling them singly is not sufficient.A new study takes a combined approach to hit two distinct molecular pathways in injured nerves and achieves substantial regrowth in an animal model.

Many lower vertebrates, like frogs and salamanders, for example, can regrow damaged nerves quite readily.And even in mammals, nerves in the periphery will regenerate and reconnect, given enough time.But nerve fibres in the brain and spinal cord do not regenerate after an injury.Researchers trying to solve this problem focused initially on figuring out what is different about the environment in the central versus the peripheral nervous system in mammals.

It was discovered early on that the myelin – the fatty sheath of insulation surrounding nerve fibres – in the central nervous system is different from that in the periphery.In particular, it inhibits nerve growth.A number of groups have tried to figure out what components of central myelin are responsible for this activity.Myelin is composed of a large number of proteins, as well as lipid membranes.One of these, subsequently named Nogo, was discovered to block nerve growth.This discovery prompted understandable excitement, especially because an antibody that binds that protein was found to promote regrowth of injured spinal nerves in the rat.(It even prompted a film, Extreme Measures, with Gene Hackman and Hugh Grant – an under-rated thriller with some surprisingly accurate science and some very serious medical malfeasance).

Unfortunately, the regrowth in rats that is promoted by blocking the Nogo protein is very limited.Similarly, mice that are mutant for this protein or its receptor show very minor regeneration.What is observed in some cases is extra sprouting of uninjured axons downstream of the spinal injury site.This can lead to some minor recovery of function but it’s really remodelling, rather than regeneration.

But it does suggest an answer to the question: why would we have evolved a system that seems actively harmful, that prevents regeneration after an injury?Well, first, the selective pressure in mammals to be able to regenerate damaged nerves is probably not very great, simply because injured animals would not typically get the chance to regenerate in the wild.And second, it suggests that the function of proteins like Nogo may not be to prevent regeneration but to prevent sprouting of nerve fibres after they have already made their appropriate connections.A lot of effort goes in to wiring the nervous system, with exquisite specificity – once that wiring pattern is established, it probably pays to actively keep it that way.

There are a number of reasons why blocking the Nogo protein does not allow nerves to fully regenerate.First, it is not the only protein in myelin that blocks growth – there are many others.Second, the injury itself can give rise to scarring and inflammation that generates a secondary barrier.And third, neurons in the mature nervous system may simply not be inclined to grow.(Not only that – the distances they may have to travel in the fully grown adult may be orders of magnitude longer than those required to wire the nervous system up during development.There are nerves in an adult human that are almost a metre long but these connections were first formed in the embryo when the distance was measured in millimetres.)

This last problem has been addressed more recently, by researchers asking if there is something in the neurons themselves that changes over time – after all, neurons in the developing nervous system grow like crazy.That propensity for growth seems to be dampened down in the adult nervous system – again, once the nervous system is wired up, it is important to restrict further growth.

Researchers have therefore looked for biochemical differences between young (developing) neurons and mature neurons that have already formed connections. The hope is that if we understand the molecular pathways that differ we might be able to target them to “rejuvenate” damaged neurons, restoring their internal urge to grow.The lab of Zhigang He at Harvard Medical School has been one of the leaders in this area and has previously found that targeting either of two biochemical pathways allowed some modest regeneration of injured neurons.(They study the optic nerve as a more accessible model of central nerve regrowth than the spinal cord).

In a new study recently published in Nature, they show that simultaneously blocking both these proteins leads to remarkably impressive regrowth – far greater than simply an additive effect of blocking the two proteins alone.The two proteins are called PTEN and SOCS3 – they are both intracellular regulators of cell growth, including the ability to respond to extracellular growth factors.The authors used a genetic approach to delete these genes two weeks prior to an injury and found that regrowth was hugely promoted.That is obviously not a very medically useful approach however – more important is to show that deleting them after the injury can permit regeneration and indeed, this is what they found.Presumably, neurons in this “grow, grow, grow!” state are either insensitive to the inhibitory factors in myelin or the instructions for growth can override these factors.

They went on to characterise the changes that occur in the neurons when these genes are deleted and observed that many other proteins associated with active growth states are upregulated, including ones that get repressed in response to the injury itself.The hope now is that drugs may be developed to target the PTEN and SOCS3 pathways in human patients, especially those with devastating spinal cord injuries, to encourage damaged nerves to regrow.As with all such discoveries, translation to the clinic will be a difficult and lengthy process, likely to take years and there is no guarantee of success.But compared to previous benchmarks of regeneration in animal models, this study shows what looks like real progress.